Competitive dCas9 binding as a mechanism for transcriptional control
Abstract Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration...
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| Format: | Article |
| Language: | English |
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Springer Nature
2021-11-01
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| Series: | Molecular Systems Biology |
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| Online Access: | https://doi.org/10.15252/msb.202110512 |
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| author | Daniel A Anderson Christopher A Voigt |
| author_facet | Daniel A Anderson Christopher A Voigt |
| author_sort | Daniel A Anderson |
| collection | DOAJ |
| description | Abstract Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27‐fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence‐independent control by adopting it for elongation control, achieving 8‐fold repression and 4‐fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis‐regulatory logic on CRISPRi‐targeted native genes. |
| format | Article |
| id | doaj-art-731a42a837d54e9783bf363e115c67f8 |
| institution | OA Journals |
| issn | 1744-4292 |
| language | English |
| publishDate | 2021-11-01 |
| publisher | Springer Nature |
| record_format | Article |
| series | Molecular Systems Biology |
| spelling | doaj-art-731a42a837d54e9783bf363e115c67f82025-08-20T02:11:49ZengSpringer NatureMolecular Systems Biology1744-42922021-11-01171111410.15252/msb.202110512Competitive dCas9 binding as a mechanism for transcriptional controlDaniel A Anderson0Christopher A Voigt1Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of TechnologySynthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of TechnologyAbstract Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27‐fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence‐independent control by adopting it for elongation control, achieving 8‐fold repression and 4‐fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis‐regulatory logic on CRISPRi‐targeted native genes.https://doi.org/10.15252/msb.202110512analog circuitCRISPRiratio sensingsynthetic biology |
| spellingShingle | Daniel A Anderson Christopher A Voigt Competitive dCas9 binding as a mechanism for transcriptional control Molecular Systems Biology analog circuit CRISPRi ratio sensing synthetic biology |
| title | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_full | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_fullStr | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_full_unstemmed | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_short | Competitive dCas9 binding as a mechanism for transcriptional control |
| title_sort | competitive dcas9 binding as a mechanism for transcriptional control |
| topic | analog circuit CRISPRi ratio sensing synthetic biology |
| url | https://doi.org/10.15252/msb.202110512 |
| work_keys_str_mv | AT danielaanderson competitivedcas9bindingasamechanismfortranscriptionalcontrol AT christopheravoigt competitivedcas9bindingasamechanismfortranscriptionalcontrol |